The long-range goal of this project is a mechanistic picture of viral entry, uncoating, replication, and assembly, derived from structure-based "molecular movies" of these processes. A large part of our research during the coming five-year period will concentrate on the molecular rearrangements that accompany penetration across a cell membrane of non-enveloped viruses - in particular, reoviruses and rotaviruses. We propose X-ray crystallographic studies of viral outer-shell proteins (u1, the reovirus penetration protein; VP4, the rotavirus binding and penetration protein; VP7, the rotavirus glycoprotein), crystallographic studies of rotavirus inner capsid particles, and biochemical studies of the reovirus u1 conformational changes that accompany penetration. We will combine structures of proteins and protein complexes with images from electron cryomicroscopy of intact virus particles, to obtain "hybrid" models of structures and intermediates that do not crystallize. We anticipate that studies of the rotavirus outer-shell proteins will facilitate design of recombinant vaccines. We will also expand our studies of the reovirus and rotavirus polymerase complexes. We can obtain a direct picture of transcription and replication, because multiple rounds of nucleotide addition can occur within crystals of the reovirus polymerase, lambda3. Finally, we will extend our structural studies of the human papillomavirus capsid protein to analyze full-sized virus-like particles, which are candidate immunogens in current vaccine trials.